An "antigen" (Ag) is any substance that will provoke an immune response. In general, antigens are considered greater than 10,000 in molecular weight and are proteins, carbohydrates or glycoproteins. Smaller, less rigid molecules are not normally antigenic in pure form, but can be made so by linking them to larger molecules. These smaller molecules are called "haptens."
Antibodies ("Ab") are immunoglobulin molecules (serum proteins) of which there are several classes. Each class has its own characteristic molecular size, electrophoretic migration velocity, carbohydrate content, etc.
Antibodies can be broken into fragments, the Fab fragment and the Fc fragment. The Fab fragment, like intact antibodies, can bind antigenic substances (e.g. haptens and antigens).
For purposes of this disclosure, a "ligand" is any chemical compound the presence or absence of which can be determined by the use of a chemical assay. With such a definition, a ligand may be an antigen, a hapten, an antibody, an antibody fragment, chemicals which can react with a cell receptor (e.g. acetylcholine), an enzyme, chelatable metals, lectins, etc. Through a chemical assay, ligands complex with some complementary chemical receptor (e.g. an antibody will bind to a specific antigen) and can then be examined to determine the presence, absence, or quantity of the ligands.
Specific chemical assay in complex mixtures generally depends on the use of specific, high affinity binding agents. These high affinity agents, e.g. antibodies, membrane receptors, enzymes, lectins, or chelates, have high binding constants. Generally, the greater the binding constant of the binding agent, the greater the chemical assay's ultimate sensitivity. Such assays are "one-shot" measurements. In the case of an immunoassay, one takes a sample, mixes the reagents, takes a reading, and discards everything. W. P. Collins, editor, Alternative Immunoassays, Wiley, 1985.
Considerable interest exists in developing specific chemical sensors, i.e. detectors which respond to changes in concentration of a specific chemical--either continuously or at least semi-continuously. Andrade, Van Wagenen, et al. "Remote Fiber-Optic Biosensors Based on Evanescent-Excited Fluoro-Immunoassay: Concept and Progress," IEEE Trans. Elect Dev., p. 1175-1179, 1985. In building such a device, a high binding constant for maximal sensitivity is desired, but a fast response time is also needed to permit continuous or semi-continuous measurements. Means for decreasing the binding constant between the reagents between measurements would be desirable. Ideally, one would "zero" the sensor between measurements and still measure the solution often enough to have a nearly continuous readout. Andrade, Van Wagenan, et al., supra. Finally, a sensor having the maximum possible dynamic range would be preferred.
The aforementioned characteristics, i.e. high binding constant, fast response time, and means for decreasing the binding constant between measurements are generally considered mutually exclusive, absent regulation of the binding constant. Andrade, Lin, et al., "Fiber Optic Immunodetectors: Sensors or Dosimeters?" SPIE 718 (1986) 280.
The high binding constants of the high affinity binding agents give them their exquisite sensitivity, but also render them with a very slow dissociation rate. Pecht, "Dynamic Aspects of Antibody Function," in M. Sela, editor, The Antigens, Vol. 6, Academic Press, 1982, page 1. Systems using such agents are in effect dosimeters rather than sensors due to this slow dissociation rate. To reuse these systems some means to weaken the bond between the binding agent (i.e. chemical receptor) and the antigenic substance (i.e. ligand) is needed to permit the complex to dissociate within a reasonable time. Andrade, Lin, et al., supra, and Lin and Schultz, IEEE Trans. Biomed. Eng. BME-33 (1986) 133.
In the case of membrane receptors for biochemicals (e.g. toxins and drugs), the ligand-receptor complex is often internalized or otherwise "turned over." Regeneration of the membrane surface in such a manner requires complex synthetic machinery which is presently impractical for man-made remote sensors.
Heretofore, the standard means to dissociate antigen-antibody (Ag-Ab) complexes is to induce a significant conformational change in Ag, Ab, or both by drastic changes in the local solution environment. These changes include radically altering the pH (e.g. to pH 2-3 or pH 11); adding to the tested solution a high concentration of chaotropic salts or a high concentration of agents which diminish hydrophobic interactions; or radically changing the temperature. Walters, "Affinity Chromatography," Anal. Chem. 57 (1985) 1099A; Parikh and Cuatrecasas, "Affinity Chromatography," Chem. Eng. News, (Aug. 26, 1985) 17; Pecht, supra; Goding, Monoclonal Antibodies, Academic Press, 1983, pp. 199-203.
Unfortunately, such treatments often lead to irreversible conformational changes which destroy the desired specific binding properties of the binding agent. Goding, supra.
In addition, it is difficult to deliver the necessary eluting agents (e.g. acids, bases or salts) on command to a remote sensor site. A different approach is to use low affinity antibodies, this approach also results in low sensitivity. Another method is to use special monoclonal antibodies with a binding site structure very susceptible to moderate changes in local pH. Hill, "Switch Immunoaffinity Chromatography with Monoclonal Antibodies," Biotechniques (1984) 14.
The high binding affinities of antibodies can lead to an almost irreversible binding of ligands to the antibodies. A need exists for a method of removing specifically bound ligands from a sensor surface without the use of damaging eluting agents. In vivo, a protein-ligand complex is usually broken down by proteolysis. In immunoassays and affinity chromatography applications, it is usually necessary to dissociate the protein-ligand complex without denaturing the protein. Thus, the dissociation of the protein-ligand complex is a fundamental problem in both basic and applied research. It would be desirable to modulate the affinity of the protein in a predictable and reversible manner.
The ability to regulate ligand-receptor binding constants would be of major significance in the development of (a) effective specific binding sensors with optimal sensitivity, response time, and dynamic range, (b) better molecular separation processes as compared to the prior art, and (c) better understanding and control of a wide range of biological and medical processes.
Certain "sensors" are described in the following patents:
U.S. Pat. No. 4,582,809 to Block, et al. discloses a method and apparatus for fluorescent immunoassay which utilizes total internal reflection to produce an evanescent wave in the liquid base. An optical fiber is provided with a plurality of coupling sites to which may be bound haptens, antigens, antibodies, and antibody fragments for reaction with a liquid to be assayed.
U.S. Pat. No. 4,558,014 to Hirschfeld, et al, discloses an apparatus and method similar to Block, et al., with "moieties of an antibody-antigen complex" (e.g. haptens, antigens, antibodies and antibody fragments.)
EPO 103 426 AZ to Hirschfeld discloses an immunoassay apparatus and method consisting of a short length of precise diameter capillary tubing having an axially disposed optical fiber to which is immobilized a monolayer of a moiety of an antibody-antigen complex, an inert diluent, and a preload of a known amount of tagged complement to the immobilized component.